Anti-BCMA antibody or antigen binding fragment thereof as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and discloses an anti-BCMA antibody or an antigen binding fragment thereof as well as a preparation method and application thereof. The amino acid sequences of the heavy chain variable regions CDR1-3 of the anti-BCMA antibody or the antigen binding fragment thereof provided by the invention are respectively shown as SEQ ID NO: 2-4, and the amino acid sequences of the light chain variable regions CDR1-3 are respectively shown as SEQ ID NO: 6-8. The anti-BCMA antibody or the antigen binding fragment thereof has excellent affinity for the BCMA protein, especially an extracellular fragment, can specifically recognize and bind the BCMA protein well, and can be used as a detection antibody of the BCMA protein to realize non-diagnostic purpose detection of the BCMA; meanwhile, the anti-BCMA antibody or the antigen binding fragment thereof can also effectively kill tumor cells overexpressing BCMA, such as pfeffer cells, through complement-dependent cytotoxicity, the cell killing rate is up to 99%, and the anti-BCMA antibody or the antigen binding fragment thereof can be used as an inhibitor of abnormal bone marrow cells overexpressing BCMA protein, is a very potential active ingredient for resisting malignant tumors of a blood system, and has broad application prospects. Good application prospects are realized.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to an anti-BCMA antibody or an antigen-binding fragment thereof, and a preparation method and application thereof. Background Art

[0002] BCMA, or tumor necrosis factor receptor superfamily member 17 (TNFRSF17), also known as B cell maturation antigen (CD269), plays a key role in promoting B cell survival and regulating humoral immune responses. BCMA is mainly expressed on the surface of mature B lymphocytes and plasma cells and is a marker protein for B lymphocyte maturation. The agonist ligands of BCMA include proliferation-inducing ligand (APRIL) and B cell activating factor (BAFF), which are mainly secreted in the bone marrow by bone marrow stromal cells, osteoclasts and macrophages in a paracrine manner. BCMA is highly correlated with multiple hematological malignancies such as multiple myeloma. BCMA overexpressed in abnormal bone marrow cells binds to agonist ligands, which leads to overactivation of multiple signaling pathways such as NF-κβ, RAS / MAPK and PI3K-PKB / Akt in bone marrow cells, thereby overexpressing anti-apoptotic proteins, cell adhesion molecules, angiogenic factors and immunosuppressive molecules, further enhancing the growth, proliferation and spread of abnormal bone marrow cells. Moreover, BCMA has similar expression levels in different stages of multiple myeloma, suggesting that BCMA has the potential to serve as an effective therapeutic target for hematological malignancies.

[0003] Therefore, obtaining antibodies with excellent specific recognition and binding capabilities for BCMA is of great significance for the development of immunotherapy for various hematological malignancies such as multiple myeloma. Summary of the invention

[0004] In the first aspect, the present invention provides an anti-BCMA antibody or an antigen-binding fragment thereof, in which the amino acid sequences of the heavy chain variable region CDR1 to 3 are shown as SEQ ID NOs: 2 to 4, respectively, and the amino acid sequences of the light chain variable region CDR1 to 3 are shown as SEQ ID NOs: 6 to 8, respectively, and the anti-BCMA antibody or the antigen-binding fragment thereof has excellent specific recognition and binding ability for BCMA.

[0005] Furthermore, in the anti-BCMA antibody or antigen-binding fragment thereof, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 5 or a variant sequence that has at least 90% homology to the amino acid sequence shown in SEQ ID NO: 5 and retains the biological activity of the corresponding parent sequence, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 9 or a variant sequence that has at least 90% homology to the amino acid sequence shown in SEQ ID NO: 9 and retains the biological activity of the corresponding parent sequence.

[0006] Furthermore, in the anti-BCMA antibody or antigen-binding fragment thereof, the amino acid sequence of the heavy chain constant region is as shown in SEQ ID NO: 27 or a variant sequence that has at least 80% homology to the amino acid sequence shown in SEQ ID NO: 27 and retains the biological activity of the corresponding parent sequence, and the amino acid sequence of the light chain constant region is as shown in SEQ ID NO: 28 or a variant sequence that has at least 80% homology to the amino acid sequence shown in SEQ ID NO: 28 and retains the biological activity of the corresponding parent sequence.

[0007] Furthermore, the anti-BCMA antibody or antigen-binding fragment thereof comprises a signal peptide having a sequence as shown in SEQ ID NO: 26, and the signal peptide is connected to the N-terminus of the heavy chain variable region and / or the N-terminus of the light chain variable region.

[0008] Furthermore, the anti-BCMA antibody or antigen-binding fragment thereof is selected from one or more of Fab, Fab', F(ab')2, Fd, Fv, dAb, single-chain antibody, chimeric antibody, multispecific antibody, monoclonal antibody and polyclonal antibody.

[0009] In a second aspect, the present invention also provides a nucleic acid molecule encoding the above-mentioned anti-BCMA antibody or antigen-binding fragment thereof.

[0010] In a third aspect, the present invention also provides a cell line that expresses and secretes the above-mentioned anti-BCMA antibody or its antigen-binding fragment.

[0011] In a fourth aspect, the present invention also provides a method for preparing the above-mentioned anti-BCMA antibody or its antigen-binding fragment, which specifically comprises: culturing the above-mentioned cell line under suitable conditions, and recovering the anti-BCMA antibody or its antigen-binding fragment from the cell culture.

[0012] In a fifth aspect, the present invention also provides the use of the above-mentioned anti-BCMA antibodies or antigen-binding fragments thereof, nucleic acid molecules and / or cell lines in BCMA detection for non-diagnostic purposes.

[0013] In a sixth aspect, the present invention also provides the use of the above-mentioned anti-BCMA antibody or antigen-binding fragment thereof, nucleic acid molecule and / or cell line in the preparation of an inhibitor of abnormal bone marrow cells.

[0014] In a seventh aspect, the present invention further provides an anti-tumor drug, which comprises the above-mentioned anti-BCMA antibody or its antigen-binding fragment, nucleic acid molecule and / or cell line.

[0015] Beneficial effects:

[0016] The anti-BCMA antibody or antigen-binding fragment thereof provided in the present invention has excellent affinity for BCMA protein, especially the extracellular segment thereof, can specifically recognize and bind to BCMA protein in different states, and can be used as a detection antibody for BCMA protein to achieve non-diagnostic detection of BCMA; at the same time, the anti-BCMA antibody or antigen-binding fragment thereof can also effectively kill tumor cells overexpressing BCMA, such as Pfeiffer cells, through complement-dependent cytotoxicity, with a tumor cell killing rate of up to 99%, and can be used as an inhibitor of abnormal bone marrow cells overexpressing BCMA protein, and is a highly potential active ingredient for anti-hematological malignancies, with good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a graph showing the experimental results of the antibody titer test of the anti-BCMA antibody provided in Example 1 of the present invention;

[0018] Figure 2 This is a graph showing the experimental results of testing the affinity of the anti-BCMA antibody to tumor cells provided in Example 2 of the present invention;

[0019] Figure 3 This is one of the experimental result graphs of the test (concentration) of the anti-BCMA antibody's ability to kill tumor cells provided in Example 2 of the present invention;

[0020] Figure 4 Figure 2 is the experimental result of testing the killing ability (concentration) of the anti-BCMA antibody on tumor cells provided in Example 2 of the present invention;

[0021] Figure 5 This is a graph showing the experimental results of testing the ability of the anti-BCMA antibody provided in Example 2 of the present invention to kill tumor cells (incubation time). DETAILED DESCRIPTION

[0022] The nucleotide sequences and amino acid sequences involved in the present invention are specifically shown in Table 1.

[0023] Table 1.

[0024]

[0025]

[0026]

[0027]

[0028]

[0029]

[0030] The present invention provides an anti-BCMA antibody or an antigen-binding fragment thereof, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:5, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:9.

[0031] In the present invention, the CDR1-3 on the heavy chain variable region of the anti-BCMA antibody or antigen-binding fragment thereof is paired with the CDR1-3 on the light chain variable region, and folded to form an antigen binding site of the BCMA extracellular segment protein, which has good binding ability with the BCMA extracellular segment protein and can specifically recognize and bind to free BCMA protein or BCMA protein on bone marrow cells. The amino acid sequences of the heavy chain variable region CDR1-3 are shown in SEQ ID NOs: 2-4, respectively, and the amino acid sequences of the light chain variable region CDR1-3 are shown in SEQ ID NOs: 6-8, respectively.

[0032] It should be noted that in the non-CDR1-3 region of the heavy chain variable region of the anti-BCMA antibody or its antigen-binding fragment, the change of some amino acid sequences has a certain effect on the binding ability of the antibody to the BCMA extracellular segment protein, but the effect is relatively small compared to the change of amino acids in the CDR region. Therefore, variant sequences that have at least 90% homology with the amino acid sequence shown in SEQ ID NO: 5 and retain the biological activity of the corresponding parent sequence are also covered within the scope of protection of the present invention. In some specific embodiments, the homology of the above variant sequence to the amino acid sequence shown in SEQ ID NO: 5 can be 90%, 92.5%, 95%, 97.5%, 99.9999% or any value therebetween.

[0033] It should be noted that in the non-CDR1-3 region of the light chain variable region of the anti-BCMA antibody or its antigen-binding fragment, the change of some amino acid sequences has a certain effect on the binding ability of the antibody to the BCMA extracellular segment protein, but the effect is relatively small compared to the change of amino acids in the CDR region. Therefore, variant sequences that have at least 90% homology with the amino acid sequence shown in SEQ ID NO: 9 and retain the biological activity of the corresponding parent sequence are also covered within the scope of protection of the present invention. In some specific embodiments, the homology of the above variant sequence to the amino acid sequence shown in SEQ ID NO: 9 can be 90%, 92%, 94%, 96%, 98%, 99.9999% or any value therebetween.

[0034] In the present invention, the amino acid sequence of the heavy chain constant region of the anti-BCMA antibody or antigen-binding fragment thereof is preferably as shown in SEQ ID NO: 27 or a variant sequence having at least 80% homology to the amino acid sequence shown in SEQ ID NO: 27 and retaining the biological activity of the corresponding parent sequence, and the amino acid sequence of the light chain constant region is preferably as shown in SEQ ID NO: 28 or a variant sequence having at least 80% homology to the amino acid sequence shown in SEQ ID NO: 28 and retaining the biological activity of the corresponding parent sequence.

[0035] In the present invention, the anti-BCMA antibody or antigen-binding fragment thereof preferably further includes a signal peptide having a sequence as shown in SEQ ID NO: 26, and the signal peptide is connected to the N-terminus of the heavy chain variable region and / or the N-terminus of the light chain variable region.

[0036] In the present invention, the type of the anti-BCMA antibody or its antigen-binding fragment may specifically be, but is not limited to, one or more of: Fab, F(ab')2, Fd, Fv, dAb, single-chain antibody, chimeric antibody, monoclonal antibody and polyclonal antibody.

[0037] It should be noted that Fab refers to the fragment with antigen binding ability obtained after the antibody molecule is hydrolyzed by papain, specifically the fragment obtained by SS bonding between the heavy chain variable region and the light chain variable region. F(ab')2 refers to the macromolecular fragment obtained by pepsin hydrolysis of the antibody molecule, specifically the fragment obtained by SS bonding between two Fabs of the heavy chain variable region, which has bivalent antibody activity and can bind to two corresponding BCMA protein antigenic determinants. The Fd fragment refers to the smallest antibody fragment containing complete antigen recognition and antigen binding sites, and the Fv fragment contains a dimer formed by a heavy chain variable region domain and a light chain variable region domain in a tight non-covalent binding manner, and the heavy chain variable region domain and the light chain variable region domain fold to produce 6 hypervariable loops, and the amino acid residues on the hypervariable loops give antigen-antibody binding specificity. The dAb fragment is a domain antibody, which is the smallest known antigen binding fragment in an antibody and can be prepared by culturing microbial cells. It has good biological activity and stability.

[0038] Based on the purpose of obtaining the above-mentioned anti-BCMA antibody or antigen-binding fragment thereof, the present invention also provides a nucleic acid molecule encoding the above-mentioned anti-BCMA antibody or antigen-binding fragment thereof.

[0039] In the present invention, the nucleic acid molecule specifically includes nucleotide fragments encoding the light chain and heavy chain of the anti-BCMA antibody or its antigen-binding fragment. The specific nucleotide sequence of the heavy chain encoding gene is shown in SEQ ID NO: 29 or is a nucleotide fragment that is completely complementary to the sequence shown in SEQ ID NO: 29 or hybridizes under moderate to high stringency conditions. The specific nucleotide sequence of the light chain encoding gene is shown in SEQ ID NO: 30 or is a nucleotide fragment that is completely complementary to the sequence shown in SEQ ID NO: 30 or hybridizes under moderate to high stringency conditions.

[0040] Based on the purpose of obtaining the above-mentioned anti-BCMA antibody or its antigen-binding fragment, the present invention also provides a cell line, which can secrete and express the above-mentioned anti-BCMA antibody or its antigen-binding fragment.

[0041] Based on the purpose of obtaining the above-mentioned anti-BCMA antibody or its antigen-binding fragment, the present invention also provides a method for preparing an anti-BCMA antibody or its antigen-binding fragment, which specifically comprises: culturing the above-mentioned cell line under suitable conditions, collecting the cell culture, and obtaining the anti-BCMA antibody or its antigen-binding fragment after purification.

[0042] Based on the excellent affinity of the above-mentioned anti-BCMA antibodies or antigen-binding fragments thereof for BCMA protein, the present invention also provides the use of the above-mentioned anti-BCMA antibodies or antigen-binding fragments thereof, nucleic acid molecules and / or cell lines in BCMA detection for non-diagnostic purposes.

[0043] Based on the excellent killing ability of the above-mentioned anti-BCMA antibodies or antigen-binding fragments thereof on abnormal bone marrow cells overexpressing BCMA protein, the present invention also provides the use of the above-mentioned anti-BCMA antibodies or antigen-binding fragments thereof, nucleic acid molecules and / or cell lines in the preparation of inhibitors of abnormal bone marrow cells.

[0044] Based on the potential of the above-mentioned anti-BCMA antibody or its antigen-binding fragment to be used as an inhibitor of abnormal bone marrow cells, the present invention also provides an anti-tumor drug, which comprises the above-mentioned anti-BCMA antibody or its antigen-binding fragment, nucleic acid molecule and / or cell line.

[0045] Embodiments of the present invention are described in detail below, and the examples of the embodiments are intended to be used to explain the present invention, but should not be construed as limiting the present invention. In the embodiments, if specific techniques or conditions are not specified, the techniques or conditions described in the literature in this area or the product specifications are used. If the manufacturer is not specified for reagents or instruments used, they are all conventional products that can be obtained commercially.

[0046] The reagents and their sources involved in the following examples specifically include:

[0047] Recombinant human BCMA extracellular segment / hFc fusion protein (Jiangsu Baiying, catalog number B528001 20201230A42); water-soluble adjuvant (Jiangsu Baiying, catalog number BMA1001); recombinant human BCMA extracellular segment-His protein (Jiangsu Baiying, catalog number B21846208 / 20230210ZA05); coating buffer (Shanghai Yuanye Biotechnology Co., Ltd., catalog number R20934); blocking solution (Shanghai Yuanye Biotechnology Co., Ltd., catalog number R22840); diluent (Shanghai Yuanye Biotechnology Co., Ltd., catalog number R40019); Goat Anti-Mous IgG-HRP (Shanghai Sigma, catalog number XGK3996); colorimetric solution (Beijing Meikowande, catalog number 1001); stop solution (Biyuntian, catalog number P025); 1640 culture medium (Sigma, catalog number R8758); FBS (Sigma, catalog number F8687); Biotin-Ter119 (BioLgend, catalog number 116204); PE fluorescent labeled antigen Recombinant Human X / His Protein (Jiangsu Baiying, catalog number B21846208); Alexa 647Goatanti-mouse IgG (BioLegend, Catalog No. 405322); Bioiotron Human IgG1 plasmid (Jiangsu Baiying, Catalog No. BIHH1X); CHO-S cells (Gibco, Catalog No. A29129); Anti-human Fc specific HRP (SouthernBiotech, Catalog No. 2040-05); Anti-human IgG-PE (Biolegend, Catalog No. 410708); Aqua Fixable Dead Cell Stain Kit (Thermo Fisher, Cat. No. L34965).

[0048] Example 1

[0049] This example is used to illustrate a method for constructing an anti-BCMA antibody. In this method, mice are immunized with a human BCMA extracellular segment protein including an amino acid fragment as shown in SEQ ID NO: 1 as an antigen, and the steps of serum antibody titer determination, cell sorting, sequencing, and humanized recombinant expression are performed, as shown below:

[0050] 1. Mouse immunization: The above-mentioned recombinant human BCMA extracellular segment / hFc fusion protein was mixed with a water-soluble adjuvant and then subcutaneously injected into mice for the first immunization. Ten days after the first immunization, 60 μg of recombinant human BCMA extracellular segment / hFc fusion protein was mixed with a water-soluble adjuvant and then subcutaneously injected into mice for the second immunization. Twelve days after the second immunization, 60 μg of recombinant human BCMA extracellular segment / hFc fusion protein was mixed with a water-soluble adjuvant and then subcutaneously injected into mice for the third immunization. The mouse serum antibody titer was detected 6 days after the third immunization.

[0051] 2. Mouse serum antibody titer determination: (1) Dilute the recombinant human BCMA extracellular segment-His protein to 2 μg / mL with coating buffer, add 100 μL to each well, adsorb and coat at 37°C for 1 h, wash the plate 6 times, and pat dry;

[0052] (2) Add 200 μL of blocking solution to each well and incubate in a 25°C incubator for 1 h. Wash the plate 6 times and pat dry.

[0053] (3) Dilute the mouse serum with blocking solution at a dilution multiple of 1:1000, and perform 10 gradient dilutions with blocking solution at a dilution multiple of 2. Add 100 μL of mouse serum obtained after the final dilution to each well, and use the mouse serum before immunization as a negative control, and the diluent as a blank control. Incubate in a 25°C incubator for 1 h, wash the plate 6 times, and pat dry.

[0054] (4) Dilute Goat Anti-Mous IgG-HRP 10,000 times with PBS, add 100 μL of diluted Goat Anti-Mous IgG-HRP to each well, incubate in a 25°C incubator for 1 h, wash the plate 6 times, and pat dry;

[0055] (5) Add 100 μL of color development solution to each well. After color development for 3 min at room temperature in the dark, add 50 μL of stop solution to each well to terminate the reaction. Read the result at 450 nm using a microplate reader.

[0056] 3. B cell sorting and sequencing: (1) Select the mouse with the highest antibody titer, remove the spleen and bone marrow, grind the spleen and bone marrow, add 1640 medium, filter with a mesh, centrifuge at 1000 r / min to remove the supernatant, add a certain volume of 1640 medium to resuspend the cells, and take Biotin-Ter119 and remove red blood cells from the solution according to the instructions to obtain a single cell suspension;

[0057] (2) Take PE fluorescently labeled antigen Recombinant Human X / His Protein and Alexa 647Goat anti-mouse IgG and the single cell suspension was co-incubated and stained according to the instructions. After washing twice, BD FACS Melody TM M flow cytometry was used to sort B cells and obtain B cells that were double positive for IgG and PE antigens;

[0058] (3) A single-cell cDNA library was constructed based on the obtained IgG and PE antigen double-positive B cells, and the cDNA library was sequenced and analyzed by NGS. The obtained antibodies and their sequences are shown in Table 2.

[0059] Table 2.

[0060]

[0061] 3. Humanized recombinant expression of antibodies: (1) Codon optimization was performed based on the amino acid sequences of each antibody shown in Table 2 to obtain the coding sequences of the heavy chain variable region and light chain variable region of each antibody, which were respectively connected to the signal peptide coding gene and the human IgG1 constant region coding gene on the Bioiotron Human IgG1 plasmid to construct a recombinant plasmid capable of expressing the corresponding humanized chimeric antibody light chain or heavy chain. The specific coding genes are shown in Table 3.

[0062] Table 3.

[0063] name sequence Signal peptide SEQ ID NO:26 Human IgG1 CH domain SEQ ID NO:27 Human IgG1 CL domain SEQ ID NO:28 B-52 antibody heavy chain encoding gene SEQ ID NO:29 B-52 antibody light chain encoding gene SEQ ID NO:30 B-96 antibody heavy chain encoding gene SEQ ID NO:31 B-96 antibody light chain encoding gene SEQ ID NO:32 B-181 antibody heavy chain encoding gene SEQ ID NO:33 B-181 antibody light chain encoding gene SEQ ID NO:34

[0064] (2) After transfecting CHO-S cells with the recombinant plasmid according to the instructions, the cells were cultured at 37° C., 115 rpm and 8% CO 2 for 7 days, and the supernatant products were collected for affinity column purification to obtain humanized chimeric antibodies, i.e., anti-BCMA antibodies.

[0065] 4. Anti-BCMA antibody titer determination: (1) Dilute the human BCMA extracellular segment protein to 2 μg / mL with coating buffer, add 100 μL to each well, adsorb and coat overnight at 4°C, wash the plate, pat dry, add 200 μL / well of PBS containing 5% skim milk powder, incubate in a 37°C incubator for 30, wash the plate 3 times, and pat dry;

[0066] (3) Anti-BCMA antibody was diluted with PBST buffer, and anti-BCMA antibody with final concentrations of 10 nM, 1 nM, 0.1 nM, 0.01 nM, 0.001 nM, 0.0001 nM, and 0.00001 nM was added to each well, and PBST buffer was used as a blank control. The plate was incubated in a 37°C incubator for 1 h, and the plate was washed 3 times and patted dry;

[0067] (4) Dilute Anti-human Fc specific HRP 16000 times with PBST buffer, add 100 μL of diluted Anti-human Fc specific HRP to each well, incubate in a 37°C incubator for 30 min, wash the plate 5 times, and pat dry;

[0068] (5) Add 100 μL of colorimetric solution to each well. After color development at room temperature and away from light for 3 min, add 50 μL of stop solution to each well to terminate the reaction. Read the result at 450 nm using an ELISA reader. Figure 1 shown.

[0069] Depend on Figure 1 It can be seen that compared with the anti-BCMA antibodies B-96 and B-181, the anti-BCMA antibody B-52 has a very good affinity for the BCMA protein.

[0070] Example 2

[0071] This example is used to illustrate the killing ability of the anti-BCMA antibodies provided in the above examples on cells that highly express BCMA. The test specifically includes:

[0072] 1. Affinity of anti-BCMA antibody to tumor cells with high BCMA expression: 2×10 5 Human diffuse large cell lymphoma B lymphocytes (Pfeiffer cells) with high BCMA expression were added to the flow tube, centrifuged at 300g for 5 minutes, and the supernatant was removed; 100 μL of anti-BCMA antibody solution with a concentration gradient (1nM, 5nM, 10nM, 25nM, 50nM and 100nM) were added to the flow tube, and incubated at 4°C for 30 minutes; 2mL of PBS was added to the flow tube, centrifuged at 300g for 5 minutes, and the supernatant was removed. Then, Anti-human IgG-PE was diluted 100 times with PBS buffer, and 100 μL of diluted Anti-human IgG-PE was added to each tube, and incubated at 4°C for 30 minutes; 2mL of PBS was added to the flow tube, centrifuged at 300g for 5 minutes, and the supernatant was removed; then 500 μL of PBS was used to resuspend, and the fluorescence signal of each tube was detected by flow cytometry, and a concentration curve was drawn. The results are as follows: Figure 2 And as shown in Table 3.

[0073] Table 3.

[0074] Monoclonal antibodies Median effective dose (nM) B-52 Antibody 8.104 B-96 Antibody 28.300 B-181 Antibody 24.980

[0075] Depend on Figure 2 From the test results shown in Table 3, it can be seen that compared with the anti-BCMA antibodies B-96 and B-181, the anti-BCMA antibody B-52 has a very good affinity for pfeiffer cells that highly express BCMA, and its half-effective dose is only 8.104 nM.

[0076] 2. Anti-BCMA antibody killing ability of tumor cells: (1) Pfeiffer cells were diluted to 5×10 5 pcs / 100 μL, and anti-BCMA antibody was added to make the final concentration of the solution antibody 0.1nM, 0.25nM, 0.5nM, 1nM, 5nM, 10nM, 25nM and 50nM, and incubated in a cell culture incubator at 37°C and 5% CO2 for 2h; 2mL of PBS was added to the flow tube, centrifuged at 300g for 5min, the supernatant was removed, and then Aqua Fixable Dead Cell Staining Kit was used to resuspend the cells according to the instructions, and incubated at room temperature for 30 minutes away from light; 2 mL of PBS was added to the flow tube, centrifuged at 300 g for 5 minutes, and the supernatant was removed; then 500 μL of PBS was used to resuspend the precipitate, and the fluorescence signal of each tube was detected by flow cytometry. The results are as follows: Figure 3 and 4 shown.

[0077] Depend on Figure 3 and 4 The test results shown in the figure show that compared with anti-BCMA antibodies B-96 and B-181, anti-BCMA antibody B-52 can efficiently kill Pfeiffer cells through complement-dependent cytotoxicity, and its IC 50 The value is 0.3903nM, which has excellent ability to kill Pfeiffer tumor cells.

[0078] (2) Pfeiffer cells were diluted to 5×10 using 1640 medium containing 25% human serum. 5 pcs / 100 μL, and added B-52 antibody to make the final concentration of the solution antibody 10 nM, and incubated in a cell culture incubator at 37°C and 5% CO2 for 2h, 4h, 6h, 8h and 12h; 2mL of PBS was added to the flow tube, centrifuged at 300g for 5min, the supernatant was removed, and then Aqua Fixable Dead Cell Staining Kit was used to resuspend the cells according to the instructions, and incubated at room temperature for 30 minutes away from light; 2 mL of PBS was added to the flow tube, centrifuged at 300 g for 5 minutes, and the supernatant was removed; then 500 μL of PBS was used to resuspend the precipitate, and the fluorescence signal of each tube was detected by flow cytometry. The results are as follows: Figure 5 shown.

[0079] From the test results shown in Table 5, it can be seen that after the anti-BCMA antibody B-52 provided by the present invention was co-incubated with Pfeiffer tumor cells for 12 hours, the killing rate of Pfeiffer tumor cells was as high as 99%, and it had very excellent tumor cell killing ability.

[0080] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention without departing from the principles and intent of the present invention.

Claims

1. An anti-BCMA antibody or an antigen-binding fragment thereof, characterized in that: In the anti-BCMA antibody or antigen-binding fragment thereof, the amino acid sequences of the heavy chain variable region CDR1 to 3 are shown in SEQ ID NOs: 2 to 4, respectively, and the amino acid sequences of the light chain variable region CDR1 to 3 are shown in SEQ ID NOs: 6 to 8, respectively.

2. The anti-BCMA antibody or antigen-binding fragment thereof according to claim 1, characterized in that: In the anti-BCMA antibody or antigen-binding fragment thereof, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 5 or a variant sequence that has at least 90% homology to the amino acid sequence shown in SEQ ID NO: 5 and retains the biological activity of the corresponding parent sequence, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 9 or a variant sequence that has at least 90% homology to the amino acid sequence shown in SEQ ID NO: 9 and retains the biological activity of the corresponding parent sequence.

3. The anti-BCMA antibody or antigen-binding fragment thereof according to claim 1, characterized in that: In the anti-BCMA antibody or antigen-binding fragment thereof, the amino acid sequence of the heavy chain constant region is as shown in SEQ ID NO: 27 or a variant sequence having at least 80% homology to the amino acid sequence shown in SEQ ID NO: 27 and retaining the biological activity of the corresponding parent sequence, and the amino acid sequence of the light chain constant region is as shown in SEQ ID NO: 28 or a variant sequence having at least 80% homology to the amino acid sequence shown in SEQ ID NO: 28 and retaining the biological activity of the corresponding parent sequence; Optionally, the anti-BCMA antibody or antigen-binding fragment thereof comprises a signal peptide whose sequence is shown in SEQ ID NO: 26, and the signal peptide is connected to the N-terminus of the heavy chain variable region and / or the N-terminus of the light chain variable region.

4. The anti-BCMA antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The anti-BCMA antibody or antigen-binding fragment thereof is selected from one or more of Fab, F(ab')2, Fd, Fv, dAb, single-chain antibody, chimeric antibody, monoclonal antibody and polyclonal antibody.

5. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the anti-BCMA antibody or antigen-binding fragment thereof according to any one of claims 1 to 4.

6. A cell line, characterized in that The cell line expresses and secretes the anti-BCMA antibody or antigen-binding fragment thereof according to any one of claims 1 to 4.

7. The method for preparing the anti-BCMA antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, characterized in that: The cell line of claim 6 is cultured under appropriate conditions, and the anti-BCMA antibody or antigen-binding fragment thereof is recovered from the cell culture.

8. Use of the anti-BCMA antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, the nucleic acid molecule according to claim 5 and / or the cell line according to claim 6 in BCMA detection for non-diagnostic purposes.

9. Use of the anti-BCMA antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, the nucleic acid molecule according to claim 5 and / or the cell line according to claim 6 in the preparation of an inhibitor of abnormal bone marrow cells.

10. An anti-tumor drug, characterized in that: The anti-tumor drug comprises the anti-BCMA antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, the nucleic acid molecule according to claim 5 and / or the cell line according to claim 6.

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